The long-term goal of the proposed research is to understand the developmental processes that regulate organ formation using radial organization of the Arabidopsis root as a model. Root radial organization is formed by three different developmental strategies. Primary roots employ a stem cell strategy, embryonic roots use a sequential division strategy and lateral roots are formed by cell proliferation from differentiated tissue. These three diverse strategies result in roots with nearly identical radial organization. A molecular-genetic approach was used to identify genes that regulate root radial organization. The SCARECROW(SCR) and SHORTROOT(SHR) genes regulate a key asymmetric division required for radial organization during all three strategies. The sequence of the SCARECROW gene indicates that it may be a transcription factor. SCARECROW is expressed within the mother cell prior to the asymmetric division and in only one of the daughter cells after the division. Its expression pattern also suggests a role during the early stages of primordia establishment. Potential upstream regulators and downstream targets of SCARECROW have been identified. The role of SCARECROW and SHORT-ROOT in the regulation of root radial organization will be characterized as a paradigm for understanding organogenesis in plants. Comparison to similar studies in animals will address the origins of shared developmental processes. The specific objectives are to: 1. Determine the molecular mechanism by which SCR regulates the asymmetric division that generates cortex and endodermis. Antibodies will be used to localize SCR and downstream and upstream regulators of SCR activity will be identified. The role of SCR during primordia establishment will be characterized by phenotypic analysis and by analysis of expression of SCR in embryonic root mutants. 2. Use the cloned SCR gene as a molecular tool to test alternative hypotheses as to how radial organization information is transmitted in embryonic, primary and lateral roots. Tissue-specific and inducible expression, sector analysis, and laser ablation will be used to determine the developmental requirements for SCR activity and how radial organization information is transmitted during root development. 3. Determine the role of SHORT-ROOT in regulating radial organization and endodermal specification. The SHORT-ROOT gene will be isolated and its interaction with the SCARECROW-mediated division path way will be characterized. 4. isolate and characterize additional alleles and mutants in root radial organization. Additional alleles and new mutants will be identified using an optical clearing method to screen for radial organization mutations.